EP1939219A1

EP1939219A1 - Polysaccharides derivatised with citric acid

Info

Publication number: EP1939219A1
Application number: EP06425873A
Authority: EP
Inventors: Marco Bosco; Luca Stucchi; Fabrizio Picotti; Rita Gianni
Original assignee: Sigea SRL
Current assignee: Sigea SRL
Priority date: 2006-12-29
Filing date: 2006-12-29
Publication date: 2008-07-02
Also published as: ATE481425T1; CA2673953A1; EP2094734B1; US8629252B2; WO2008081257A3; PL2094734T3; PT2094734E; SI2094734T1; DE602007009306D1; EP2094734B9; AU2007341080A1; JP5372774B2; WO2008081257A2; US20100311687A1; ES2348215T3; JP2010514879A; EP2094734A2; DK2094734T3

Abstract

Derivatives of oligo/polysaccharides with the formula

wherein:
X is OH, O-M, NH-R₁, O-R₁;
M is an alkaline or alkaline-earth metal, transition metal, or cation containing a quaternary nitrogen atom;
Y is H or R₂;
R1: the residue of an oligo/polysaccharide;
R2: the residue of a C1-C4 linear chain aliphatic carboxylic acid or citric acid.

Description

This invention relates to oligo/polysaccharides characterised in that they possess ester or amide bonds with citric acid, are not crosslinked in relation to the starting saccharide, and are soluble in water. The ester or amide bonds involve the carboxyl functions of citric acid and the hydroxyl or amino functions present on the starting oligo/polysaccharide.
In view of the bland, controllable conditions needed to obtain them, these derivatives have well-defined, reproducible characteristics and do not present any further structural or molecular-weight modifications compared with the starting oligo/polysaccharide.
They have a very high hydratability capacity per unit of weight and consequently, in the hydrated state, possess a high capacity for hydrating systems external to their contact, such as the skin systems or mucous membranes. These derivatives also manifest a modulatable ability to complex/salify metal ions such as Ag, Zn, Fe, Cu, etc.. In view of these characteristics, and especially their constant composition and reproducibility, the products according to the invention can be advantageously used in the pharmaceutical and cosmetic industries as hydrating agents or constituents of pharmaceutical compositions, or as complexes/salts of metal ions such as Ag, Zn, Fe or Cu in the healing of indolent sores. The complexes/salts can also be used as bacteriostatic/antibacterial agents, immunostimulants and antisideropenic agents.
The invention also relates to the process for their production in aqueous solution, in water/solvent, or in organic solvent only, but preferably in organic solvent. The reaction conditions, at very bland temperatures, do not degrade the saccharide, and oligo/polysaccharides which are homogenous in terms of degree of substitution are obtained. Moreover, the citrate residue can be esterified on the hydroxyl with a C1-C4 linear-chain aliphatic carboxylic acid or with citric acid. The esters to the hydroxyl obtained constitute a further subject of the invention.

Prior art

Processes are known wherein starch and cellulose are reacted with citric derivatives (citric acid or citric anhydride). These processes basically comprise the following steps: 1) formation of a paste or suspension of polysaccharide and citric acid, containing little or no water, in the presence of agents potentially able to induce the formation of the intermediate esterifying citric agent (generally considered to be citric anhydride), by mixing for preset periods of time; 2) removal of water until the mixture is dry; 3) heating of the dry product at high temperatures (up to 180°C). These stages, especially drying and heat treatment, are liable to cause extensive degradative structural changes in the initial polysaccharides (demolition of the saccharide chains with reduction of molecular weight, oxidation and elimination) and final polysaccharides (random, uncontrolled intermolecular crosslinking, etc.), and do not guarantee the constant composition and reproducibility of the final materials. However, as stated below, the use of the products obtained is designed for fields of application in which these requirements are not industrially crucial, or particularly required in regulatory terms. It is sufficient for them to possess average characteristics (e.g. a metal-ion sequestering capacity, liquid-absorbing capacity, etc.) which are technologically acceptable, even if they vary within wide ranges; above all, they must involve low manufacturing costs, as the applications are designed for a market involving very large quantities but low added value. This latter aspect also explains why citric acid is used rather than citric anhydride, which is far more expensive, preferring to opt for its formation in situ by citric acid (with all the associated problems of composition and reproducibility mentioned above).
The uses proposed for these products are as follows:

1. as food additives, due to their ability to prevent syneresis in frozen foods, or as dietary fibre;
2. as a heavy-metal sequestering resin in the treatment of waste water, or simply as a biodegradable ion-exchange resin.

Thus, for example, US 2,461,139 describes the synthesis of starch derivatives using citric anhydride obtained in situ from citric acid and acetic anhydride; both a dry method and a method in alkaline aqueous suspension are used. The derivatives obtained can be used in the textile, paper and food industries.
US 2,935,510 describes the synthesis process of acetic and propionic esters of starch in aqueous suspensions, using citric acid as crosslinking agent. These derivatives are used as additives in frozen foods.
Numerous references in the literature describe the process of derivatisation of starch with citric acid by the dry method at the temperature of 110-140°C (Starch, 30, 1978, No. 2, pp. 47-51); these studies demonstrate that the dry process requires very precise control of the reaction parameters (temperature, etc.) to prevent excessive crosslinking (Starch, 48,1996, No. 7/8, pp. 275-279). The dry process does not involve a high degree of substitution; DS values (the ratio between moles of citrate residues and moles of polysaccharide) of between 12.2% and 14.4% (Starch, 51, 1999, No. 10, pp. 354-361) and 16.0% (Starch, 56, 2004, pp. 364-370) are reported in the literature. Due to the persistence of the polysaccharide at high temperatures, the dry process causes partial degradation of the polymer chain and the formation of by-products (Starch, 54, 2002, pp. 185-192).
Some patents relate to derivatisation of cellulose or wood with citric acid.
US 2,759,787 describes a dry process for the synthesis of cellulose derivatives which produces a polymer matrix insoluble in water and organic solvents; the product obtained can be used as a resin that sequesters large molecules or ions in aqueous solution. The use of citric acid as a crosslinking agent for other polysaccharides is also reported:

1. hydroxypropylmethylcellulose (Carb. Pol, 51, 2003, pp. 265-271) for the production of mechanically resistant films;
2. chitosan cross-linked with wool fibres (J. Appl. Polym. Sci, 94, 2004, pp. 1999-2007) to obtain fabrics with antimicrobial properties;
3. β-cyclodextrins bonded to chitosan with a citrate bridge to obtain products with antimicrobial activity.

Many of the patents and articles cited presuppose the formation of citric anhydride, obtained by dehydration of citric acid by the action of heat (in dry processes) or following treatment with suitable desiccant agents.
EP 282289 reports the synthesis process and cosmetic use of a salt of a citric acid monoester esterified with long-chain aliphatic alcohols. Monoesters of citric acid in which the alcohol derives from carbohydrates (oligo- or polysaccharides) are not cited.
In conclusion, derivatives of citric acid with starch and cellulose are already known in the literature, while chitosan has been used to bond cyclodextrins esterified with citric acid, which acts as a bifunctional bridge.
The known products are obtained by the dry method from crude polysaccharide and citric acid at approx. 150-180°C, or in basic aqueous slurries from crude starch, citric acid and the anhydride of an organic acid. However, the products obtained by these methods present a low degree of substitution, as defined above (maximum DS = 16%), and the polymer undergoes degradation effects due to the reduction of molecular weight and the formation of double bonds on the glucose rings, or other collateral reactions. These effects are often unimportant for a product designed, for example, as a flocculant/sequestering agent of metal ions in waste water. The synthesis conditions are therefore designed to promote crosslinking, in order to enhance these properties.
Esters of polysaccharides with citric acid are therefore needed which have well-defined, reproducible characteristics, and whose structure is not radically different from the natural polysaccharide, so as to extend their application possibilities.

Description of the invention

This invention relates to non-crosslinked, highly citrated, water-soluble polysaccharides which possess unexpected hygroscopicity, and are useful as constituents of cosmetic or pharmaceutical formulations.
The polysaccharides according to the invention do not present irreversible structural alterations in the saccharific component (the prerequisite for pharmaceutical and cosmetic applications) as their synthesis involves very bland conditions (ambient temperature, an inert solvent such as formamide or DMF, and activation by triethylamine under apparent pH conditions of between 6.5 and 9.0), which are therefore not degradative.
The process according to the invention also includes the synthesis and isolation of cyclical citric anhydrides wherein the hydroxyl can be esterified by known methods with C1-C4 linear-chain aliphatic carboxylic acids (formic, acetic, trifluoroacetic, dichloroacetic, trichloroacetic, propionic or butyric acid). These derivatives are then reacted with oligo/polysaccharides to give products characterised by ester or amide bonds with citric acid, absence of crosslinking and solubility in water. The ester or amide bonds involve the carboxyl functions of citric acid and the hydroxyl or amino functions present on the starting oligo/polysaccharide.
The direct use of cyclic citric anhydride under the bland conditions described guarantees that in the products obtained, only one carboxyl of citric acid is bonded to the saccharide residue, while the other two are present in acid or salified form.
The derivatives according to the invention have the following formula:
wherein:

X is OH, O^-M, NH-R₁, O-R₁;
M is an alkaline or alkaline-earth metal, transition metal, or cation containing a quaternary nitrogen atom;
Y is H, R₂;
R1: the residue of an oligo/polysaccharide;
R2: the residue of a C1-C4 linear-chain aliphatic carboxylic acid or citric acid.

The oligo/polysaccharides can be chosen from among cellulose, amylose, starch, scleroglucan, curdlan, laminaran, pullulan, gellan, agar, carrageenans, chitin, chitosan, hyaluronic acid, heparin and other glycosaminoglycans, mannans, galactans, galactomannans, glucomannans, konjac, locust bean gum, gum acacia, gum tragacanth, guarans, acemannan and their derivatives.
The following are particularly preferred:

starch fractions with a high amylose content or amylose fractions with a low molecular weight (dextrins);
soluble derivatives of alkylcellulose such as carboxymethylcellulose, hydroxyethylcellulose and hydroxypropylcellulose
chitosan with different molecular weights and different percentages of residual N-acetyl-glucosamine.
Said oligo/polysaccharides typically have a molecular weight of between 10³ and 10⁷ Daltons.

The process for the preparation of the products according to the invention involves the addition of a solution containing cyclic citric anhydride or cyclic citric anhydride esterified to the hydroxyl with a C1-C4 linear-chain aliphatic carboxylic acid or citric acid, or a mixture of said citric anhydrides and a base with a solution of oligo-polysaccharide in a suitable organic solvent (formamide, dimethylformamide, NMP (N-methylpyrrolidone) or dimethylsulphoxide).
Examples of bases are organic bases containing one atom of trisubstituted nitrogen, which may be aliphatic (e.g. triethylamine, DBO, DBU, DABCO or hexamine), aromatic (e.g. imidazole, pyridine or dimethylaminopyridine) or heterocyclic (e.g. pyrrolidine), an inorganic base (e.g. K₃PO₄, K₂HPO₄ or M_nCO₃, with M=alkaline or alkaline-earth metal), or a mixture thereof. Preferred bases are triethylamine, dimethylaminopyridine (DMAP), imidazole or pyridine.
The products according to the invention present a degree of substitution in citrate ester (or citrate starch) of between 0.01 and 1.00 compared with the repetitive unit of the saccharide, and preferably between 0.16 and 0.50.
The products according to the invention present the carboxyls in acid form or in the form of salts of alkaline or alkaline-earth metals, transition metals (such as Zn, Cu and Ag). or cations with quaternary nitrogen atoms.
The products according to the invention can be used in pharmaceutical formulations as additives for moisturising cosmetic formulations, skin care and personal hygiene, or as medical aids with a disinfectant or antibacterial action, etc., possibly suitably formulated with cationic antibiotics or antifungals.
The following examples illustrate the invention in greater detail.

Examples:

The ¹H NMR analyses are conducted in D₂O by Bruker Avance 400 spectrometer equipped with a 5 mm multinuclear probe with gradient z, at 300°K. The analyses also use diffusion-ordered experiments (DOSY: Diffusion Ordered Spectroscopy).

Example 1. Synthesis of starch citrate ester

5.0 g of starch was solubilised in 167 ml of formamide at 90°C for 3 hours; the temperature was then reduced to 25°C. 3.8 g of citric anhydride, dissolved in 7 ml of formamide, and 12.5 ml of triethylamine were added. The reaction was maintained under agitation for 16 hours at 25°C. 300 ml of water was then added, and the mixture was purified by ultrafiltration. The aqueous solution was then frozen and freeze-dried. 5.2 g of lyophilisate was recovered.
10 mg of lyophilisate was solubilised in 0.7 ml of D₂O and transferred to an NMR analysis tube. A DS value of 28% was obtained from integration of the methylene signals associated with citric acid (at 2.8 ppm).

Example 2. Synthesis of carboxymethylcellulose citrate ester

5.0 g of carboxymethylcellulose sodium salt was solubilised in 165 ml of formamide at 95°C for 5 hours; the temperature was then reduced to 25°C. 3.9 g of citric anhydride, dissolved in 30 ml of formamide, and 15.0 ml of triethylamine were added. The reaction was maintained under agitation for 6 hours at 25°C. 200 ml of water was then added, and the mixture was purified by ultrafiltration. The aqueous solution was then frozen and freeze-dried. 5.3 g of lyophilisate was recovered.
10 mg of lyophilisate was solubilised in 0.7 ml of D₂O and transferred to an NMR analysis tube. A DS value of 23% was obtained from integration of the methylene signals associated with citric acid (at 2.8 ppm).

Example 3. Synthesis of chitosan starch and citrate ester

316 mg of chitosan was solubilised in 35 ml of water acidified with trifluoroacetic acid at pH 3, and then freeze-dried. 457 mg of lyophilisate was recovered and redissolved in 23 ml of formamide at ambient temperature. 121 mg of citric anhydride, dissolved in 2 ml of formamide, and 230 µl of triethylamine were added. The reaction was maintained under agitation for 16 hours at 25°C. 30 ml of water was then added, and the mixture was purified by dialysis. The aqueous solution was then frozen and freeze-dried. 240 mg of lyophilisate was recovered.
10 mg of lyophilisate was solubilised in 0.7 ml of D₂O and transferred to an NMR analysis tube. A DS value of 29% was obtained from integration of the methylene signals associated with citric acid (at 2.8 ppm).

Example 4. Synthesis of pullulan citrate ester

125 mg of pullulan starch was solubilised in 4 ml of formamide at 80°C for 15 minutes; the temperature was then reduced to 25°C. 121 mg of citric anhydride, dissolved in 1.5 ml of formamide, and 430 µl of triethylamine were added. The reaction was maintained under agitation for 16 hours at 25°C. 30 ml of water was then added, and the solution was neutralised to pH 7. Finally, the mixture was purified by ultrafiltration. The aqueous solution was then frozen and freeze-dried. 157 mg of lyophilisate was recovered.
10 mg of lyophilisate was solubilised in 0.7 ml of D₂O and transferred to an NMR analysis tube. A DS value of 36% was obtained from integration of the methylene signals associated with citric acid (at 2.8 ppm).

Example 5. Synthesis of hyaluronic acid citrate ester

200 mg of hyaluronic acid sodium salt was solubilised in 6.6 ml of formamide at 80°C for 4 hours; the temperature was then reduced to 25°C. 87 mg of citric anhydride, dissolved in 1.0 ml of formamide, and 278 µl of triethylamine were added. The reaction was maintained under agitation for 16 hours at 25°C. 100 ml of water was then added, and the solution was neutralised to pH 7. Finally, the mixture was purified by dialysis and ultrafiltration. The aqueous solution was then frozen and freeze-dried. 235 mg of lyophilisate was recovered.
10 mg of lyophilisate was solubilised in 0.7 ml of D₂O and transferred to an NMR analysis tube. A DS value of 18% was obtained from integration of the methylene signals associated with citric acid (at 2.8 ppm).

Example 6. Synthesis of dextrin citrate ester

105 mg of dextrin 10 was solubilised in 4 ml of formamide at 25°C; 112 g of citric anhydride, dissolved in 1.5 ml of formamide, and 460 µl of triethylamine were added. The reaction was maintained under agitation for 4 hours at 25°C. The reaction mixture was then acidified with TFA and dripped into acetone under energetic agitation. The precipitate obtained was decanted, centrifuged and washed twice with 10 ml of acetone, centrifuged again, and finally dried.
10 mg of lyophilisate was solubilised in 0.7 ml of D₂O and transferred to an NMR analysis tube. A DS value of 27% was obtained from integration of the methylene signals associated with citric acid (at 2.8 ppm).

Claims

Derivatives of oligo/polysaccharides with the formula
wherein:
X is OH, O^-M, NH-R₁, O-R₁;

M: is an alkaline or alkaline-earth metal, transition metal, or cation containing a quaternary nitrogen atom;

Y is H or R₂;

R1: the residue of an oligo/polysaccharide;

R2: the residue of a C1-C4 linear chain aliphatic carboxylic acid or citric acid.
Derivatives as claimed in claim 1, wherein R1 is a residue of cellulose, amylose, starch, scleroglucan, curdlan, laminaran, pullulan, gellan, agar, carrageenans, chitin, chitosan, hyaluronic acid, heparin and other glycosaminoglycans, mannans, galactans, galactomannans, glucomannans, konjac, locust bean gum, gum acacia, gum tragacanth, guarans, acemannan or their derivatives.
Derivatives as claimed in claim 2, wherein R1 is a starch residue with a high content of amylose or amylose fractions with a low molecular weight (dextrins).
Derivatives as claimed in claim 2, wherein R1 is a residue of carboxymethylcellulose, hydroxyethylcellulose or hydroxypropylcellulose.
Derivatives as claimed in claim 2, wherein R1 is a residue of chitosan with different percentages of residual N-acetyl-glucosamine.
Derivatives according to any of claims 1 to 5, having a molecular weight of between 10³ and 10⁷ Daltons, and preferably between 10⁴ and 5x10⁵ Daltons.
Derivatives according to any of claims 1 to 6, having a degree of substitution in citrate ester or citrate starch of between 0.01 and 1.00 x N, where N is equal to the number of hydroxyls contained in the repetitive unit of the saccharide.
Derivatives as claimed in claim 7, having a degree of substitution in citrate ester or citrate starch of between 0.16 and 0.50 compared with the repetitive unit of the saccharide.
Derivatives according to any of claims 1 to 8, wherein the carboxyls are present in acid form or in the form of salts of alkaline or alkaline-earth metals, transition metals or cations with atoms of quaternary nitrogen.
Process for the preparation of the derivatives claimed in claims 1-9, which involves the addition of a solution containing cyclic citric anhydride or cyclic citric anhydride esterified to the hydroxyl with a C1-C4 linear-chain aliphatic carboxylic acid or citric acid, or a mixture of said citric anhydrides and a base with a solution of oligo-polysaccharide in a suitable organic solvent.
Process as claimed in claim 10, wherein the solvent is formamide, dimethylformamide, N-methylpyrrolidone or dimethylsulphoxide.
Process as claimed in claim 10 or 11, wherein the base is an aliphatic, aromatic or heterocyclic organic base containing one atom of trisubstituted nitrogen, an inorganic base or a mixture thereof.
Process as claimed in claim 12, wherein the base is chosen from among triethylamine, dimethylaminopyridine, imidazole or pyridine.
Use of the derivatives claimed in claims 1-9 as additives for moisturising cosmetic formulations, skin care and personal hygiene.
Pharmaceutical compositions or medical aids comprising the derivatives claimed in claims 1-9, possibly mixed with suitable excipients, vehicles or active ingredients.